Transcript Slide 1

In the name of
God
Summer School
Influenza Unit,
Pasteur Institute of Iran
summer 2010
Basics of Cell Culture
Introduction
• Cell culture is the process by which
prokaryotic, eukaryotic or plant cells are
grown under controlled conditions. But in
practice it refers to the culturing of cells
derived from animal cells.
• Cell culture was first successfully undertaken
by Ross Harrison in 1907
Historical events in the development of cell culture
• 1878: Claude Bernard proposed that physiological systems of an
organism can be maintained in a living system after the death .
• 1880: Roux maintained embryonic chick cells in a saline culture.
• 1911: Lewis made the first liquid media consisted of sea water,
serum, embryo extract, salts and peptones. They observed limited
monolayer growth.
Contd..
• 1916: Rous and Jones introduced proteolytic enzyme trypsin for
the subculture of adherent cells.
• 1923: Carrel and Baker developed 'Carrel' or T-flask as the first
specifically designed cell culture vessel.
• 1940s: The use of the antibiotics penicillin and streptomycin in
culture medium decreased the problem of contamination in cell
culture.
• 1952: Gey established a continuous cell line from a human cervical
carcinoma known as HeLa (Helen Lane) cells. Dulbecco developed
plaque assay for animal viruses using confluent monolayers of
cultured cells.
History
Aseptic techniques
Carrel Flask
Contd..
• 1955: Eagle studied the nutrient requirements of selected cells in
culture and established the first widely used chemically defined
medium.
• 1965: Ham introduced the first serum-free medium which was able
to support the growth of some cells.
• 1978: Sato established the basis for the development of serumfree media from cocktails of hormones and growth factors.
Major development’s in cell culture technology
• First development was the use of antibiotics which inhibits the
growth of contaminants.
• Second was the use of trypsin to remove adherent cells to
subculture further from the culture vessel.
• Third was the use of chemically defined culture medium.
Why is cell culture used for?
Areas where cell culture technology is currently playing a major
role.
• Model systems for Studying basic cell biology, interactions between disease causing
agents and cells, effects of drugs on cells, process and triggering of aging & nutritional
studies.
• Toxicity testing , Study the effects of new drugs.
• Cancer research, Study the function of various chemicals, virus & radiation to convert
normal cultured cells to cancerous cells.
Contd….
• Virology
Cultivation of virus for vaccine production, also used to study there
infectious cycle.
•
Genetic Engineering
Production of commercial proteins, large scale production of viruses
for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B &
measles
•
Gene therapy
Cells having a functional gene can be replaced to cells which are
having non-functional gene
Terminology
Primary Cell Culture
• Derived from an explant, directly from the animal
• Usually only survive for a finite period of time
• Involves enzymatic and/or mechanical disruption of the
tissue and some selection steps to isolate the cells of
interest from a heterogeneous population
Clone
• A population derived from a single cell
Sub-culture
• Transplantation of cells from one vessel to another
Established or Continuous Cell Lines
• A primary culture that has become immortal due to
some transformation
• Most commonly tumour derived, or transformed with a
virus such as Epstein-Barr
Primary culture
• Cells when surgically or enzymatically removed from an organism and
placed in suitable culture environment will attach and grow are called
as primary culture
• Primary cells have a finite life span
• Primary culture contains a very heterogeneous population of cells
• Sub culturing of primary cells leads to the generation of cell lines
• Cell lines have limited life span, they passage several times before they
become senescent
• Cells such as macrophages and neurons do not divide in vitro so can be
used as primary cultures
• Lineage of cells originating from the primary culture is called a cell
strain
Continous cell lines
• Most cell lines grow for a limited number of generations after which
they ceases
• Cell lines which either occur spontaneously or induced virally or
chemically transformed into Continous cell lines
• Characteristics of continous cell lines
-smaller, more rounded, less adherent with a higher
nucleus
/cytoplasm ratio
-Fast growth and have aneuploid chromosome number
-reduced serum and anchorage dependence and grow more in
suspension conditions
-ability to grow upto higher cell density
-different in phenotypes from donar tissue
Types of cells
On the basis of morphology (shape & appearance) or on
their functional characteristics. They are divided into
three.
• Epithelial like-attached to a substrate and appears
flattened and polygonal in shape
• Lymphoblast like- cells do not attach remain in
suspension with a spherical shape
• Fibroblast like- cells attached to an substrate appears
elongated and bipolar
Common cell lines
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Human cell lines
-MCF-7
breast cancer
HL 60
Leukemia
HEK-293
Human embryonic kidney
HeLa
Henrietta lacks
Primate cell lines
Vero
African green monkey kidney epithelial cells
Cos-7
African green monkey kidney cells
And others such as CHO from hamster, sf9 & sf21 from insect
cells
Monkey Kidney cells
Polio vaccine first product primary monkey kidney
cells human diploid lung fibroblast
Common cell lines
BHK
Fibroblast
Baby hamster kidney
CHO
Epithelial
Chinese Hamster Ovary
PER-C6
Epithelial
Human Keratonocyt
MDCK
Epithelial
Canine Kidney
Vero
Fibroblast
Monkey Kidney
3T3
Fibroblast
Mouse fibroblast
HeLa Cells
• Classic example of an
immortalized cell line.
– These are human epithelial cells from a
fatal cervical carcinoma transformed by human
papillomavirus 18 (HPV18).
HeLa Cells
• Adherent cells which maintain contact inhibition in vitro:
– As they spread out across the culture flask, when two
adjacent cells touch, this signals them to stop growing.
• Loss of contact inhibition is a classic sign of oncogenic cells:
– Cells which form tumors in experimental animals.
– Such cells not only form a monolayer in culture but also pile up on
top of one another.
• HeLa cells are not oncogenic in animals, but they may become so if
further transformed by a virus oncogene.
Number of Cell Divisions
• Growing cells in culture.
– Place cells in a culture dish.
– Give them nutrients, growth
factors, keep them free from
bacterial.
– Cells will grow to cover the
surface of the dish.
– Can take cells out of this culture
and start a new culture.
• Splitting cells from one dish to
another is a passage.
Number of Cell Divisions
• This ability to split cells and
have them continue to divide is not
without limits however.
• Normal cells have a limit to
the number of times which
they can be passed in culture.
• This number does vary from cell
type to cell type, but commonly the
limit is between 50 and 100
passages.
Contact Inhibition
• The phenomenon observed in normal animal cells that causes them
to stop dividing when they come into contact with one another.
• Cells in a culture flask with the appropriate nutrients and the cells
grow and divide.
• Continues until the cells are covering the entire surface.
• At that point they stop dividing.
• These cells can be triggered to begin dividing again by giving
them more room.
• The cells now being in an environment where they are not in
contact with one another begin to divide again.
Contact Inhibition
• Cancer cells do not display contact inhibition.
• Put them in a culture dish, they will grow to create a
single layer of cells
• Then they will continue to grow multiple layers and
create piles of cells.
GROWTH CYCLE IN ATTACHEMENT
CULTURE
• Eukaryotic cells in attachment culture have a characteristic growth cycle
similar to bacteria.
• The growth cycle is typically divided into three phases.
– Lag Phase
– Log Phase
– Plateau Phase
Lag Phase
• This is the time following subculture and reseeding during which
there is little evidence of an increase in cell number.
• It is a period of adaptation during which the cell replaces
elements of the glycocalyx lost during trypsinization, attaches to
the substrate, and spreads out.
• During spreading the cytoskeleton reappears and its reappearance
is probably an integral part of the spreading process.
Log Phase
• This is the period of exponential increase in cell number following
the lag period and terminating one or two doublings after
confluence is reached.
• The length of the log phase depends on the seeding density, the
growth rate of the cells, and the density at which cell proliferation
is inhibited by density.
• In the log phase the growth fraction is high (usually 90%-100%) and
the culture is in its most reproducible form.
It is the optimal time for sampling since the population is at its most
uniform and viability is high.
• The cells are, however, randomly distributed in the cell cycle and, for
some purposes, may need to be synchronized.
Plateau Phase
• Toward the end of the log phase, the culture becomes confluent
– All the available growth surface is occupied and all the cells are
in contact with surrounding cells.
• Following confluence the growth rate of the culture is reduced,
and in some cases, cell proliferation ceases almost completely
after one or two further population
doublings.
• At this stage, the culture enters the plateau (or stationary)
phase, and the growth fraction falls to between 0 and10%.
• There may be a relative increase in the synthesis of
specialized versus structural proteins and the constitution
and charge of the cell surface may be changed.
Culture media
• Choice of media depends on the type of cell being cultured
• Commonly used Medium are GMEM, EMEM,DMEM etc.
• Media is supplemented with antibiotics viz. penicillin, streptomycin
etc.
• Prepared media is filtered and incubated at 4 C
Culture Media
• Ions
– Na, K, Ca, Mg, Cl, P, Bicarbonate
• Trace elements
– iron, zinc, selenium
• Sugars
– glucose is the most common
• Amino acids
– 13 essential
• Vitamins
• Serum
– Contains a large number of growth promoting activities such as
buffering toxic nutrients by binding them, neutralizes trypsin and other
proteases
– Contains peptide hormones or hormone-like growth factors that
promote healthy growth.
• Antibiotics - although not required for cell growth, antibiotics are
often used to control the growth of bacterial and fungal
contaminants.
Serum
• Serum/protein free media
– Growth factors
– Lipid concentrate
– Extracts Yeast extract
– Insulin
– Bovine Serum Albumin ( transport/detoxification)
Basic equipments used in cell culture
• Laminar cabinet-Vertical are preferable
• Incubation facilities- Temperature of 25-30 C for insect & 37 C for
mammalian cells, co2 2-5% & 95% air at 99% relative humidity. To
prevent cell death incubators set to cut out at approx. 38.5 C
• Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin) &
media components (e.g. glutamine & serum) at -20 C
• Microscope- An inverted microscope with 10x to 100x magnification
• Tissue culture ware- Culture plastic ware treated by polystyrene
Equipment
Class II Cabinets
These cabinets are designed to
give operator protection as well
as a sterile environment
The air is directed downwards
from the top of the cabinet to the
base, when working in these
cabinets it is important not to
pas non-sterile objects over
sterile ones
Equipment
Centrifuges
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There are centrifuges in each cell culture area which are
refrigerated
•
100 x g is hard enough to sediment cells, higher g forces
may damage cells
Incubators
•
The incubators run at 37C and 5% Carbon Dioxide to
keep the medium at the correct pH
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They all have meters on them to register temperature and
gas level
•
There are alarms to indicate when these deviate from set
parameters
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Keep the door open for as short a time as possible
Why sub culturing?
• Once the available substrate surface is
covered by cells (a confluent culture) growth
slows & ceases.
• Cells to be kept in healthy & in growing state
have to be sub-cultured or passaged
• It’s the passage of cells when they reach to
80-90% confluency in flask/dishes/plates
• Enzyme such as trypsin, dipase, collagenase in
combination with EDTA breaks the cellular
glue that attached the cells to the surface
Culturing of cells
• Cells are cultured as anchorage dependent or
independent
• Cell lines derived from normal tissues are considered
as anchorage-dependent grows only on a suitable
substrate e.g. tissue cells
• Suspension cells are anchorage-independent e.g. blood
cells
• Transformed cell lines either grows as monolayer or as
suspension
Adherent cells
• Cells which are anchorage dependent
• Cells are washed with PBS (free of ca & mg ) solution.
• Add enough trypsin /EDTA to cover the monolayer
• Incubate the plate at 37 C for 1-2 min.
• Tap the vessel from the sides to dislodge the cells
• Add complete medium to dissociate and dislodge the
cells
• with the help of pipette which are remained to be
adherent
• Add complete medium depends on the subculture
• requirement either to 75 cm or 175 cm flask
Suspension cells
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Easier to passage as no need to detach them
As the suspension cells reach to confluency
Asceptically remove 1/3rd of medium
Replaced with the same amount of pre-warmed
medium
Culturing Animal Tissue- the Steps
• Animal tissue is obtained
either from a particular
specimen, or from a ‘tissue
bank’ of cryo-preserved (cryo
= frozen at very low
temperatures in a special
medium)
• Establishment of the tissue is
accomplished in the required
medium under aseptic
conditions
Culture vessels and medium
for animal cell culture
Working with cryopreserved cells
• Vial from liquid nitrogen is placed into 37 C water bath, agitate vial
continuously until medium is thawed
• Centrifuge the vial for 10 mts at 1000 rpm at RT, wipe top of vial with
70% ethanol and discard the supernatant
• Resuspend the cell pellet in 1 ml of complete medium with 20% FBS
and transfer to properly labeled culture plate containing the
appropriate amount of medium
• Check the cultures after 24 hrs to ensure that they are attached to the
plate
• Change medium as the colour changes, use 20% FBS until the cells
are established
Freezing cells for storage
• Remove the growth medium, wash the cells by PBS and remove the
PBS by aspiration
• Dislodge the cells by trypsin-versene
• Dilute the cells with growth medium
• Transfer the cell suspension to a 15 ml conical tube, centrifuge at
200g for 5 min at RT and remove the growth medium by aspiration
• Resuspend the cells in 1-2ml of freezing medium
• Transfer the cells to cryovials, incubate the cryovials at -80 C overnight
• Next day transfer the cryovials to Liquid nitrogen
Cell viability
• Cell viability is determined by staining the cells with trypan
blue
• As trypan blue dye is permeable to non-viable cells or
death cells whereas it is impermeable to this dye
• Stain the cells with trypan dye and load to
haemocytometer and calculate % of viable cells
- % of viable cells= Nu. of unstained cells x 100
total nu. of cells
Cell toxicity
• Cytotoxicity causes inhibition of cell growth
• Observed effect on the morphological alteration in the cell layer or
cell shape
• Characteristics of abnormal morphology is the giant cells,
multinucleated cells, a granular bumpy appearance, vacuoles in the
cytoplasm or nucleus
• Cytotoxicity is determined by substituting materials such as medium,
serum, supplements flasks etc.
Contaminant’s of cell culture
Cell culture contaminants of two types
• Chemical-difficult to detect caused by endotoxins,
plasticizers, metal ions or traces of disinfectants that
are invisible
• Biological-cause visible effects on the culture they are
mycoplasma, yeast, bacteria or fungus or also from
cross-contamination of cells from other cell lines
Cell Culture Enemies
Micro-organisms grow ~10-50 times faster than
mammalian cells, which take ~8-16 hours to
divide. They are more tolerant to variations in
temperature, pH and nutrient supply than cells.
Cells are most vulnerable to contamination when
our aseptic technique is bad and the culture
becomes infected with bugs.
This can lead to the development of antibiotic
resistant micro-organisms.
Cell Culture Enemies
Cells are more susceptible to infection at certain times
• When they have been stressed after recovery from
liquid nitrogen
• Primary cells are often generated by enzymatic
disruption and selection procedures
• Cultures prepared from live animals will often be
accompanied by micro-organisms
• Splitting cells at too high a dilution can allow microorganisms to dominate the culture
• Cells release Autocrine growth factors which
condition the medium and favour cell growth
Cell Culture Enemies
IF YOU ARE IN DOUBT ABOUT THE CONDITION OF YOUR CELLS,
ASK FOR ADVICE
NEVER USE CONTAMINATED CELLS. THEY MAY NOT REACT IN
THE SAME WAY AS UNCONTAMINATED CELLS
POOR ASEPTIC TECHNIQUE IS THE MAJOR CAUSE OF
INFECTIONS
Effects of Biological Contamination’s
• They competes for nutrients with host cells
• Secreted acidic or alkaline by-products ceses the growth
of the host cells
• Degraded arginine & purine inhibits the synthesis of
histone and nucleic acid
• They also produces H2O2 which is directly toxic to cells
Detection of contaminants
• In general indicators of contamination are turbid culture
media, change in growth rates, abnormally high pH, poor
attachment, multi-nucleated cells, graining cellular
appearance, vacuolization, inclusion bodies and cell lysis
• Yeast, bacteria & fungi usually shows visible effect on the
culture (changes in medium turbidity or pH)
• Mycoplasma detected by direct DNA staining with
intercalating fluorescent substances e.g. Mycoplasma also
detected by enzyme immunoassay by specific antisera or
monoclonal abs or by PCR amplification of mycoplasmal
RNA
Basic aseptic conditions
• If working on the bench use a Bunsen flame to heat the
air surrounding the Bunsen
• Swab all bottle tops & necks with 70% ethanol
• Flame all bottle necks & pipette by passing very quickly
through the hottest part of the flame
• Avoiding placing caps & pipettes down on the bench;
practice holding bottle tops with the little finger
• Work either left to right or vice versa, so that all
material goes to one side, once finished
• Clean up spills immediately & always leave the work
place neat & tidy
Safety aspect in cell culture
• Possibly keep cultures free of antibiotics in order to be able to recognize the
contamination
• Never use the same media bottle for different cell lines. If caps are dropped or
bottles touched unconditionally touched, replace them with new ones
• Necks of glass bottles prefer heat at least for 60 secs at a temperature of 200 C
• Switch on the laminar flow cabinet 20 min prior to start working
• Cell cultures which are frequently used should be subcultered & stored as
duplicate strains
Safety
Use of Cell Culture areas
• The cell culture area, as any other
laboratory is a working area
• Do not bring your friends in with
you
• Do not eat, drink or smoke in these
areas
• Do wear a lab coat at all times
whether in a cell culture area or a
laboratory
• Do wear disposable gloves, but
make sure that you dispose of
them in the correct way before you
leave the area
Other key facts…….?
• Use actively growing cells that are in their log phase of
growth, which are 80-90% viable
• Keep exposure to trypsin at a minimum
• Handle the cells gently. Do not centrifuge cells at high
speed or roughly re-suspend the cells
• Feeding & sub culturing the cells at more frequent
intervals then used with serum containing conditions
may be necessary
• A lower concentration of 10 4cells/ml to initiate
subculture of rapidly growing cells & a higher
concentration of 105cells/mlfor slowing growing cells
Thanks